JPS61233694A - Growth factor contained in bovine spleen and method of isolating same - Google Patents

Abstract

PURPOSE:To obtain the titled factor suitable for making a serum-free medium, having specific molecular weight, isoelectric point, etc., by grinding bovine spleen and a salt solution, treating an extracted supernatant liquid with a weakly acidic cation exchanger and treating the resultant substance with heparin immobilized Sepharose. CONSTITUTION:A bovine spleen is ground in a salt solution (e.g., solution of salt having <= about 1.5 ms electric conductivity), an extracted solution is obtained by centrifugation, etc., subjected to salting-out with ammonium sulfate, dialyzed against 0.1M sodium phosphate buffer solution at 6.0 pH, brought into contact with a weakly acidic cation exchanger to adsorb a growth factor and a fraction containing the growth factor is eluted with 0.1M sodium phosphate buffer solution containing 0.6M salt at 6.0 pH. Then, the eluate is brought into contact with heparin immobilized Sepharose equilibrated at 7.0 pH to adsorb the growth factor, whichis eluted with a buffer solution having a high salt concentration to give the aimed growth factor having about 16,000-18,000 molecular weight, 9.5-10.5 isoelectric point and affinity for heparin immobilized Sepharose.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to growth factors contained in bovine spleen and a method for isolating the same.

Conventional technology Many animal cells are passaged using cell culture techniques.
Some of these systems, especially those derived from humans, are about to be widely applied as means for producing useful substances. Examples include trying to make monoclonal antibodies and lymphokines be produced by lymphoid cells, and interferon by fibroblasts. In many cases, the addition of serum such as fetal bovine serum or newborn bovine serum to the culture medium is essential for culturing these cells.

Problems to be Solved by the Invention However, it is also true that there are various restrictions on the cultivation, production, extraction, and purification of useful substances. One of its biggest limitations is that a large number of cells can survive or proliferate only in the presence of serum. Also, this serum is called
Isolation and purification of useful substances may become extremely difficult due to the presence in the culture medium of a mixture of proteins whose composition is not completely clear.

It is no exaggeration to say that research on serum-free media or synthetic media has become active in recent years, starting from these reasons 5. However, these studies have also hit a major wall. The biggest reason for this is that the growth factors present in serum are unknown.

In other words, it is not clear what kind of growth factors are present in serum. Here it is worth studying various growth factors. In other words, discovering, isolating, and purifying growth factors for various cells can be said to be an essential step in researching serum-free or synthetic media that can replace serum-supplemented media. To date, epidermal growth factor (
Epidermal Growth Factor; E
GF ), platelet derived growth factor (PlateletDerived) shown in U.S. Patent No. 4,979,896
Growth Factor; PDGF)
etc. have been discovered and isolated. Among these, epithelial cell growth factor requires a fairly high effective concentration of several tens of ng/ml, and platelet-derived growth factor is effective at a few ng/ml, but there are restrictions on raw materials, Yield remains a problem. Recently, Gospodarowicz et al.
Proceedings of the Nation
al Academy of 5science, 81st
Volume 6963-6967) and Lobb et al.
Chemistry, Volume 23, 6295-62
Fibroblast growth factor (Fibl
oblast Growth Factory FGF
) was isolated, and the effective concentration ranged from several + pg/nl to several n g
It was shown that the concentration was extremely low./ysl.

However, according to both reports, the fimbriae cell growth factor obtained from bovine brain, which is easily available in large quantities, is at most 50%.
Bovine pituitary gland 1, which is NG and difficult to obtain in large quantities
Only about 200 ng of fibroblast growth factor can be obtained immediately.

Means to Solve the Problem If a large amount of growth factor with the same activity as the fibroblast growth factor isolated from bovine brain or bovine pituitary gland, which was isolated by Gospodarowicz et al., could be obtained, serum-free medium or There is no doubt that this will greatly contribute to research on synthetic media. The present inventors have conventionally focused on υ fibroblast growth factor,
After extensive research, they discovered that the growth factor activity was contained in bovine spleen, which is easily available in large quantities as a raw material, and were able to isolate it.

Crush the bovine spleen with a saline solution to obtain the extracted supernatant,
This is treated with a weakly acidic cation exchanger to obtain a crude fraction containing growth factors. This is further treated with heparin-immobilized Sepharose to isolate the desired growth factor.

A feature of the present invention is the isolation of growth factors from bovine spleen extract. That is, bovine spleen extract cannot be used as it is because it contains strong growth inhibitory factors in addition to growth factors when culturing cells. As mentioned above, the bovine spleen is crushed in a saline solution (sodium chloride, ammonium sulfate, etc. with an electrical rest conductivity of about 1.5 m 8 or less), and an extract is obtained by centrifugation or the like.
This extract is salted out with ammonium sulfate 30%-75% saturated. The resulting precipitate was thoroughly dialyzed against 0.1 M sodium phosphate buffer, pH 6.0, and then diluted with a weakly acidic cation exchanger (e.g. CM-Sephadex C-50, CM -52, etc.) to adsorb the growth factors, the exchanger was washed with the buffer used for equilibration and 0.1M sodium phosphate, pH 6.0, containing 0.15M sodium chloride. 0.1M phosphoric acid containing 6M sodium chloride) IJum buffer, pH
Both fractions containing growth factors are eluted at 6.0. The obtained growth factor-containing fraction was diluted with 0.0
The growth factors were adsorbed by contacting with heparin-immobilized Sepharose equilibrated with 1M sodium phosphate buffer, pH 7.0, and the same resin was mixed with the buffer used for equilibration and 0.01M phosphate containing 1.0M sodium chloride. sodium acid, pH
After washing with 7.0 and chloride), the growth factor can be isolated by using a buffer solution with increased concentration of IJium.

In this isolation step, one or both of the steps of salting out with ammonium sulfate and subsequent dialysis may be omitted without causing any essential difference in the obtained growth factor. However, treatment with a weakly acidic cation exchanger and treatment with heparin-immobilized Sepharose is essential.

The molecular weight of the growth factor thus obtained was determined by 8D8-polyacrylamide gel electrophoresis to be approximately 16.000~
It showed a molecular weight of 18,000 and an isoelectric point of 9.5 to 10.5 by isoelectric focusing using ampholine. Growth factor activity was determined in bovine fetal heart endothelial cells (Fetal Bov).
ine Hea,t EndothelialCell
, ATCC, CRL-1395) and 3H-thymidine uptake activity using mouse 3T3 cells, even tens of pg/ml showed a promoting effect.

Effects of the Invention Comparing the growth factor activities, the effective concentration of the above-mentioned epidermal growth factor is several tens of ng/ml, and the effective concentration of platelet-derived growth factor, which is said to have strong growth factor activity, is several ng/ml.
/me, the growth factor contained in the bovine spleen of the present invention exhibits a promoting effect even at a few tens of pg/ynl and has extremely strong activity, and the scope of its application will be quite wide in the future. Seem.

Another advantage of the present invention is that the raw material, bovine spleen, is readily available in large quantities.

Examples Example 1 Beef tiles R#4 were cut into small pieces, 8 tons of 0.1jM ammonium sulfate aqueous solution was added, and the mixture was crushed using a homogenizer. After adjusting to I) H4,5 by adding phosphoric acid, the mixture was centrifuged to obtain a supernatant. Add sodium hydroxide to this supernatant to adjust the pH.
After returning to about 7, add ammonium sulfate to 30%
The mixture was saturated, centrifuged, and the precipitate was discarded. Ammonium sulfate was further added to the supernatant to achieve 75% saturation, followed by centrifugation and the supernatant was discarded. The obtained precipitate was dissolved in water and diluted with 0.1M
The mixture was thoroughly dialyzed against IJum buffer, pH 6.0. After removing the insoluble substances generated after dialysis by centrifugation, the mixture was transferred to a column (column size 3.6 x 15 crn) packed with CM-Sephadex C-50 equilibrated with 0.1 M sodium phosphate buffer, pH 6.0. After adsorption of the growth factors through the column, the same column was washed with the buffer used for equilibration and 0.1 M sodium phosphate buffer containing 0.15 M sodium chloride, I) H6,0, .6M
Contains sodium chloride trO, IM sodium phosphate, p
The growth factor fraction was eluted with H6,0.

The obtained fraction was 0.6M chloride) containing 0.6M chloride.
Growth factors were adsorbed through a column (column size 1.2X10 crn) packed with heparin-immobilized Sepharose equilibrated with 01M sodium phosphate buffer, p) (7,0), and the same column was packed with the buffer used for equilibration. liquid and 1.0M
After washing with IJum buffer, pH 7.0 (0.01M phosphoric acid containing sodium chloride), chloride from 1.0M to 2.0M based on 0.01M sodium phosphate buffer, pH 7.0. Growth factors were eluted using a linear sodium gradient elution method. The growth factor is sodium chloride concentration 1
．． It was eluted around 5M.

In the growth factor obtained by this method, only a single protein band was detected at a molecular weight of about 17,000 by 8DS-polyacrylamide gel electrophoresis. Growth factor activity was determined in bovine fetal heart endothelial cells (Fetal
Bovine Heart Endothelial
When the cell proliferation promoting activity was measured using CellXATCC, CRL-1395), the results shown in Table 1 were obtained.

Table 1 0・1 34 0.3 44 1.0 74 3.0 110 Number of implanted cells; 5 Growth factors were eluted using a stepwise elution method using .0M sodium chloride. The obtained fraction was 5D8
- When analyzed by polyacrylamide gel electrophoresis, the same results as in Example 1 were obtained, except that a few bands of extremely small amounts of inclusions were observed. The measurement results of cell proliferation promoting activity were also the same as in Example 1.

Example 3 After obtaining a 30% to 75% saturated ammonium sulfate precipitate according to Example 1, the precipitate was dissolved in water to reduce the electrical conductivity to 1.5.
CM-52, 150 ml equilibrated with 0.1 MIJ sodium disodinate buffer 6.0 after adjusting to 8 m
was added and stirred at 5°C for 1 hour. Then, after filtration on a glass filter, the same resin was washed with the buffer used for equilibration, packed into a column with a diameter of 3.6 m, and then
Washed with 0.1M sodium phosphate buffer containing 5M sodium chloride, pH 6.0. 0.1M phosphoric acid (containing 0.6M sodium chloride), IJum, pH 6.0, and both fractions containing growth factors were extracted, and the obtained fractions were subjected to heparin-immobilized Sepharose column chromatography using the method shown in Example 2. Graphography was performed to isolate the growth factors.

The same results as in Example 2 were obtained in both analysis by 8D8-polyacrylamide gel electrophoresis and measurement of cell proliferation promoting activity.

Example 4 Four pieces of bovine spleen were cut into small pieces, 8 tons of 0.05M sodium chloride was added, and the mixture was crushed using a homogenizer. The extraction supernatant was obtained in the same manner as in Example 1, and sodium hydroxide was added to the supernatant to make pns, o, and then 0.1M sodium phosphate,
CM-52,1501/ equilibrated at pH 6.0 was added and stirred at 5°C for 1 hour.

After washing according to the method shown in Example 3, the column was filled, and further washing and elution were performed. The obtained growth factor-containing fraction was subjected to heparin-immobilized Sepharose column chromatography according to the method shown in Example 1 to isolate growth factors.

The same results as in Example 1 were obtained in both analysis by 8D8-polyacrylamide gel electrophoresis and measurement of cell proliferation promoting activity.

Claims (2)

[Claims] (1) Growth factor contained in bovine spleen and having the following properties: (A) Molecular weight approximately 16,000-18,000 (B) Isoelectric point 9.5-10.5 (C) Heparin-immobilized Sepharose has a strong affinity for (2) Molecular weight contained in bovine spleen: approximately 16,000 to 1
8,000, an isoelectric point of 9.5 to 10.5, a method for isolating a growth factor comprising the following steps in the process of isolating a growth factor having an affinity for heparin-immobilized sepharose, (a) Partially purified from bovine spleen extract using a weakly acidic cation exchanger. (b) Growth factors are isolated from the growth factor-containing fraction obtained in (a) using heparin-immobilized Sepharose.